Methods for producing synthetic surfaces that mimic collagen coated surfaces for cell cultue

ABSTRACT

The present invention discloses methods for producing synthetic surfaces that mimic collagen coated surfaces for cell culture comprising: providing a monomer source comprising one or more organic compounds which are capable of polymerization, wherein at least one organic compound is prolinol; creating a plasma of said monomer source; and contacting at least a portion of a surface with the plasma to provide a plasma polymer coated surface. Advantageously, such methods provide an animal-free, synthetic, chemically defined surface that mimics a collagen coated surface for cell culture. Advantageously, such methods not only reduce the cost and/or issues associated with animal-derived collagen but are also amenable to large scale manufacturing.

FIELD OF THE INVENTION

The present invention relates to methods for producing syntheticsurfaces that mimic collagen coated surfaces as well as surfacesprepared by such methods.

BACKGROUND OF THE INVENTION

Collagen coated surfaces have been widely used in cell culture topromote attachment of fastidious cells, including primary cells such ashepatocytes and keratinocytes. Generally, collagen derived from anon-human animal (e.g., rat tail) is employed to coat surfaces for cellculture. However, the use of such collagen can be problematic, forexample, in human therapeutic applications. Although human collagen canbe used for coating such surfaces, the cost is very high. Likewise,although surfaces coated with peptide sequences that mimic collagencoated surfaces have also been made to culture cells, the cost ofproducing such surfaces is relatively high and simply not suitable forlarge scale manufacturing. Thus, there is a need for methods to produceanimal-free, synthetic, chemically defined surfaces that mimic collagencoated surfaces which are less costly than those presently available andsuitable for large scale manufacturing as well as surfaces produced bysuch methods.

SUMMARY OF THE INVENTION

The present invention discloses methods for producing animal-free,synthetic, chemically defined surfaces that mimic collagen coatedsurfaces for cell culture. Advantageously, such methods not only reducethe cost and/or issues associated with animal-derived collagen but arealso amenable to large scale manufacturing.

In particular, the present invention provides methods for producing asynthetic surface that mimics a collagen coated surface for cell culturecomprising:

i) providing a monomer source comprising one or more organic compoundswhich are capable of polymerization, wherein at least one organiccompound is prolinol;ii) creating a plasma of the monomer source; andiii) contacting at least a portion of a surface with the plasma toprovide a plasma polymer coated surface wherein the plasma polymercoated surface mimics one or more functional characteristics of acollagen coated surface.In addition, the present invention provides surfaces useful for cellculture produced by the methods described above.

The present invention also provides a surface for cell culture whereinat least a portion of the surface comprises a coating of prolinol.

The present invention further provides a surface for cell culturewherein at least a portion of the surface comprises a coating comprisinga single type of amino acid wherein the single type of amino acid isproline.

These and other features of the invention will be better understoodthrough a study of the following detailed description.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a flowchart representing a method in accordance with thesubject invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses methods for producing a syntheticsurface that mimics a collagen coated surface for cell culture.Likewise, the present invention provides surfaces useful for cellculture. Collagen is a triple helical coiled coil structure with aregular arrangement of amino acids in each of the helical unit. Thesequence often follows the pattern

Gly-Pro-Y or Gly-X-Hyp (hydroxyproline), where X and Y may be any ofvarious amino acid residues and wherein the motif Gly-Pro-Hyp occursfrequently.

Though not meant to be limited by any theory with the subject invention,a proline-like monomer fixed or immobilized to a surface may mimic oneor more functional characteristics of a collagen coated surface.Preferably, the proline-like monomer is fixed by plasma polymerization.Generally, monomers with relatively high vapor pressure are required sothat a monomer can readily be introduced into the vacuum chamber as avapor during the polymerization process. For example, monomers commonlyused in plasma polymerization, such as allylamine and acrylic acid, haverelatively high vapor pressure. In contrast, monomers with relativelylow vapor pressure, such as amino acids, although useable, are notpreferred for use in plasma polymerization.

Prolinol, a commercially available chiral amino-alcohol (e.g.,D-Prolinol is available from Sigma-Aldrich under Catalog No. 81744) is aderivative of proline. As prolinol is a liquid with greater vaporpressure than the amino acid proline, prolinol is more amenable for useas a monomer source in creating a plasma for coating a surfacetherewith. In particular, treating a surface for cell culture by plasmapolymerization of prolinol provides a synthetic surface that mimics acollagen coated surface. In fact, human hepatocytes are able to attachto a surface coated with prolinol alone without any furtherextracellular matrix protein coating.

For plasma polymerization, the cell culture vessels to be coated areloaded into a chamber of a plasma polymerization reactor. The chamber isthen pumped down to create a vacuum. The vapor of monomer sourcecomprising prolinol is introduced into the chamber. A radio-frequencypower is then turned on to initiate the polymerization of prolinol onthe surface(s) of the cell culture vessels inside the chamber.

In one embodiment, a RF excited plasma is employed for plasmapolymerization. However, any method of generating a gaseous plasma maybe used, for example a glow discharge or a corona discharge. Forexample, microwave frequencies may be employed instead of, or inaddition to, RF excitation.

In one embodiment, the plasma is a pulsed plasma. Exemplary conditionsfor plasma polymerization wherein the plasma is pulsed include, but arenot limited to, an on/off pulse of 1 ms/50 ms and an RF power of 100 W;an on/off pulse of 10 ms/100 ms and an RF power of 5 W; on/off pulse of30 ms/100 ms and an RF power of 5 W; and on/off pulse of 5 ms/50 ms andan RF power of 100 W.

In another embodiment, the plasma is a continuous wave plasma. Exemplaryconditions for plasma polymerization wherein the plasma is a continuouswave plasma include, but are not limited to, an RF power of 5 W.

Gases typically used with plasma treatment and introduced into theplasma chamber include Ar, He, Ne, He, He/H₂, O₂, N₂, NH₃, and CF₄.

In one embodiment, prolinol is deposited onto the surface by plasmapolymerization. A flowchart depicting a method for producing a syntheticsurface by plasma polymerization of prolinol is shown in FIG. 1. In oneembodiment, the surface mimics one or more functional characteristics ofa collagen coated surface. In one embodiment, human hepatocytes attachto the coating. In one embodiment, the coating consists essentially ofprolinol.

Alternative means for coating a surface with prolinol include, but arenot limited to, chemical vapor deposition or immobilization by covalentattachment to one or more carboxyl functional groups, one or more aminefunctional groups or a combination thereof. Notably, chemical vapordeposition is discussed in Dobkin and Zuraw (Dobkin and Zuraw (2003).Principles of Chemical Vapor Deposition. Kluwer). In one embodiment,prolinol is deposited onto the surface by chemical vapor deposition.

In another embodiment, prolinol is immobilized on the surface bycovalent attachment to one or more carboxyl functional groups, one ormore amine functional groups or a combination thereof. It is understoodthat for covalent attachment, the surface may require pre-activationsuch that the surface comprises one or more carboxyl functional groups,one or more amine functional groups or a combination thereof tofacilitate the binding of prolinol thereto. Exemplary means of covalentimmobilization of prolinol include, but are not limited to, providing acarboxyl functionalized surface (i.e., wherein the carboxyl groups arcactivated) using carbodiimide chemistry (e.g., EDC/NHS) followed bylinking of prolinol to such surface through an amine reaction with theNHS groups on the surface. Alternatively, covalent immobilization may beachieved by providing an aldehyde functionalized surface followed bylinking prolinol to such surface through an amine reaction with thealdehyde groups on the surface through Schiff base formation followed bystabilization of the Schiff base through sodium borohydride reduction.

Similarly, though not meant to be limited by theory with the subjectinvention, a single type of amino acid, e.g., proline, fixed orimmobilized to a surface may mimic one or more functionalcharacteristics of a collagen coated surface. Notably, proline makes upabout 9% of collagen.

In one embodiment, proline is immobilized by covalent attachment to oneor more carboxyl functional groups, one or more amine functional groupsor a combination of two or more thereof. In one embodiment, the surfacemimics one or more functional characteristics of a collagen coatedsurface. In one embodiment, human hepatocytes attach to the coating. Inone embodiment, the coating consists essentially of proline.

Exemplary means for coating a surface with proline include, but are notlimited to, covalent attachment to one or more carboxyl functionalgroups, one or more amine functional groups or a combination thereof. Itis understood that for covalent attachment, the surface may requirepre-activation such that the surface comprises one or more carboxylfunctional groups, one or more amine functional groups or a combinationthereof to facilitate the binding of proline thereto.

Similar to covalent immobilization of prolinol, exemplary means ofcovalent immobilization of proline include, but are not limited to,providing a carboxyl functionalized surface (i.e., wherein the carboxylgroups are activated) using carbodiimide chemistry (e.g., EDC/NHS)followed by linking of proline to such surface through an amine reactionwith the NHS groups on the surface. Alternatively, covalentimmobilization may be achieved by providing an aldehyde functionalizedsurface followed by linking proline to such surface through an aminereaction with the aldehyde groups on the surface through Schiff baseformation followed by stabilization of the Schiff base through sodiumborohydride reduction.

In one embodiment, the surface is a multiwell plate, a dish, or a flask.In one embodiment, the monomer source consists essentially of prolinol.

The phrase “mimics one or more functional characteristics of a collagencoated surface” as used herein with reference to a surface coated withprolinol or proline includes but is not limited to functionalcharacteristics of collagen that includes attachment of cells to acollagen coated surface. For example, the attachment of humanhepatocytes to a collagen coated surface. In one embodiment, one or morefunctional characteristics of a collagen coated surface comprisesattachment by human hepatocytes.

Example A

To explore the ability of the prolinol-coated surface to mimic one ormore functional characteristics of a collagen coated surface, humanhepatocytes were seeded and monitored on both collagen-coated andprolinol-coated surfaces under the same culture conditions. In brief,cryopreserved hepatocytes were removed from liquid nitrogen storage andimmediately placed in a 37° C. waterbath until the cells were nearlythawed. The contents were then transferred to 50 mls of pre-warmedISOM's Seeding Media. The tubes were centrifuged in a Low-speedcentrifuge at 50×g for 5 minutes at room temperature. The supernatantfluid was aspirated and discarded. The cell pellet was resuspended in1-2 mLs of ISOM's Seeding Media. Cells were counted and then diluted toa density of 10⁶ cells/ml ISOM's Seeding Media. Cells were seeded at adensity of 4×10⁵ cells/well (i.e., a volume of 400 μL/well of 10⁶cells/mL) onto a 24-well plate. Specifically, a prolinol coated orCollagen Type I coated plate. The plates were placed in an incubator at37° C. with 5% CO₂ for about 4 hrs. After such time, the ISOM's SeedingMedia was aspirated and cells were fed with 400 μL/well of HepatoSTIM™media (BD, Catalog # 355056). Hepatocyte cell attachment was observedafter 24 hours.

In addition, cell attachment and spreading on the surfaces were analyzedand microscopic images taken following several days of cell culture.Notably, human hepatocyte attachment to the prolinol-coated surface issimilar to that observed for a collagen-coated surface. Moreover, itshould be noted that human hepatocytes attached to the prolinol-coatedsurface without any further extracellular matrix protein coating.

1. A method for producing a synthetic surface that mimics a collagencoated surface for cell culture comprising: i) providing a monomersource comprising one or more organic compounds which are capable ofpolymerization, wherein at least one organic compound is prolinol; ii)creating a plasma of the monomer source; and iii) contacting at least aportion of a surface with the plasma to provide a plasma polymer coatedsurface wherein the plasma polymer coated surface mimics one or morefunctional characteristics of a collagen coated surface.
 2. The methodof Claim 1, wherein the plasma is a pulsed plasma.
 3. The. method ofClaim 1, wherein the plasma is a continuous wave plasma.
 4. The methodof Claim 1, wherein the surface is a multiwell plate, a dish, or aflask.
 5. The method of Claim 1, wherein the monomer source consistsessentially of prolinol.
 6. The method of Claim 1, wherein one or morefunctional characteristics of a collagen coated surface comprisesbinding human hepatocytes.
 7. A surface produced by the method of Claim1.
 8. A surface produced by the method of Claim
 5. 9. A surface for cellculture wherein at least a portion of the surface comprises a coating ofprolinol.
 10. The surface of Claim 9, wherein prolinol is deposited ontothe surface by plasma polymerization.
 11. The surface of Claim 9,wherein prolinol is deposited onto the surface by chemical vapordeposition.
 12. The surface of Claim 9, wherein prolinol is immobilizedon the surface by covalent attachment to one or more carboxyl functionalgroups, one or more amine functional groups or a combination thereof.13. The surface of Claim 9, wherein the surface mimics one or morefunctional characteristics of a collagen coated surface.
 14. The surfaceof Claim 9, wherein human hepatocytes attach to the coating.
 15. Thesurface of Claim 9, wherein the coating consists essentially ofprolinol.
 16. A surface for cell culture wherein at least a portion ofthe surface comprises a coating comprising a single type of amino acidwherein the single type of amino acid is proline.
 17. The surface ofClaim 16, wherein proline is immobilized by covalent attachment to oneor more carboxyl functional groups, one or more amine functional groupsor a combination of two or more thereof.
 18. The surface of Claim 16,wherein the surface mimics one or more functional characteristics of acollagen coated surface.
 19. The surface of Claim 16, wherein humanhepatocytes attach to the coating.
 20. The surface of Claim 16, whereinthe coating consists essentially of proline.